It has long been known that Agrobacterium (Agrobacterium tumefaciens), is a soil bacterium which causes Crown gall disease in many dicotyledonous plants. In the ninety-seventies, it was found that the Ti plasmid of Agrobacterium is involved in pathogenicity and that T-DNA which is part of the Ti plasmid is integrated into the plant genome. It was later revealed that the T-DNA contained the hormone synthesis genes (cytokinins and auxins) necessary for crown gall tumorigenesis and that those genes, although derived from bacteria, are expressed in plants. A group of genes that are located in the virulence region (Vir region) of the Ti plasmid are necessary to the excision of T-DNA and its transfer to plants, and furthermore, the border sequences that are located on opposite ends of T-DNA are required for the excision, which are called the right border sequence and the left border sequence. Agrobacterium rhizogenes, another Agrobacterium species has a similar system involving the Ri plasmid.
Stated more specifically, the proteins produced on the basis of the genes located in the vir region (vir proteins) recognize the right and left border sequences to integrate the T-DNA located between the border sequences into plant genome. This function provided the basis for the transformation of plants with a foreign gene pre-inserted into T-DNA, thereby giving rise to the development of Agrobacterium-mediated plant transformation technology.
Most recently, however, several reports have appeared describing that, in certain kinds of plants, it is sometimes observed that T-DNA is not excised at the border sequences, and hence, T-DNA can be transferred into the plant chromosome together with a region adjacent to T-DNA (Ramanathan et al., Plant Molecular Biology 28, 1149–1154 (1995), and Kononov et al., Plant Journal 11, 945–957 (1997)). If a DNA element other than T-DNA is co-transferred, the resulting transgenic plants will be suspected of having unexpected characteristics, which could have a negative impact on public acceptance of food products made of transgenic plants. It is therefore desired to develop a method whereby it can be ensured that unnecessary non-T-DNA sequences of Agrobacterium will not transfer to plant chromosomes.
The inventors supposed that the vir proteins of Agrobacterium sometimes fail to recognize the border sequences and this may explain the reason why non-T-DNA is transferred into plant chromosome together with T-DNA. No vectors have yet been developed that can suppress or reduce the transfer of non-T-DNA segment with a view to solving said problem.
Based on the above supposition, the inventors have conducted intensive studies for creating vectors for use in Agrobacterium-mediated transformation. In order to reduce the probability that non-T-DNA element is transferred to plant chromosome the inventors modified the vector with a view to increasing the efficiency of the vir proteins of Agrobacterium to recognize the border sequence/s. As a result, it has been found that while two border sequences exist in the transformation vector, the probability of the integration of non-T-DNAs can be reduced by providing a plurality of left border sequences. The present invention has been accomplished on the basis of this finding.